The goal of the research is to understand the function and control of DNA methylation in eukaryotes. We already know that methylation 1) can affect gene expression and 2) can cause mutations. The proposed collaboration will combine the biochemical expertise of my laboratory to dissect the mechanism and function of DNA methylation in Neurospora crassa, an organism well suited for both biochemical and genetic studies. Specific aims of the planned experiments are: 1. To define what constitutes a methylation "signal" in Neurospora. Discovery and characterization of RIP (repeat-induced point mutation) led to the conclusion that a sprinkling of G:C to A:T mutations can induced methylation. The proposed research will test a rapid in vivo assay for de novo methylation and apply it in experiments: a) to look for short sequences capable of preventing or inducing methylation of surrounding sequences, b) to determine how many G:C to A:T mutations are required to trigger methylation of a chromosomal region and which positions are critical, and c) to ascertain if various types of mutations (e.g., polarized transitions vs. transversions and frameshifts) are equally effective in triggering methylation. 2. To look for evidence of maintenance methylation in Neurospora. Evidence for cooperativity in methylation of opposite chains in Neurospora DNA will be sought and, if feasible, maintenance of methylation patterns will be directly assessed. 3. To determine whether methylated and unmethylated sequences of Neurospora are in different chromatin forms. A methyl-CpG binding protein available in the Bird laboratory will be employed to affinity-purify oligonucleosomes containing methylated sequences. This chromatin will be characterized with respect to acetylation state of histones H3 and H4 and presence of histone H1. 4. To determine whether Neurospora has a methyl-C binding protein. 5. To determine whether DNA methylation is associated with DNA replication in Neurospora.